Column for removing β2 -microglobulin

ABSTRACT

A column comprising an insoluble carrier and an anti-β 2  -microglobulin antibody immobilized to the carrier can specifically adsorb and remove β 2  -microglobulin in the blood. This column is useful for the prevention and treatment of diseases such as carpal tunnel syndrome observed in patients undergoing blood dialysis.

TECHNICAL FIELD

This invention relates to a column for removing β₂ -microglobulin. Moreparticularly, this invention relates to a column which specificallyadsorbs and removes β₂ -microglobulin in the blood.

BACKGROUND ART

β₂ -microglobulin is a light chain of a double-stranded proteinconstituting the major histocompatibility antigen (in case of human, itis HLA, class I), and occurs on the surfaces of most of cells. It alsooccurs in the body fluid in the free form, but the physiologicalfunction of the free β₂ -microglobulin has not yet been known. The fullamino acid sequence thereof has been determined for human and othervarious animals, and its three dimensional structure has been determinedby X-ray analysis for bovine. It has been proved that it is a simpleprotein with a molecular weight of about 12,000, which does not have asugar chain, and that it has structurally high homology with the Cdomain (constant domain) of immunogulobulin. Further, the homology ofthe amino acid sequence thereof between different species is 60 to 80%,and thus it is considerably high (Proc. Natl. Acad. Sci. 257, 2619(1982)).

The β₂ -microglobulin level in the blood of the patients suffering fromnephropathy, who are undergoing artificial blood dialysis for a longperiod, is as high as 10 to 100 times that of normal human. It isassumed that this is because that β₂ -microglobulin which is decomposedin the kidney is not removed by the blood dialysis and thus accumulatesin the blood.

The present inventors separated and analyzed the amyloid proteinsdeposited on the diseased part of a patient suffering from carpal tunnelsyndrome to find that most of the amyloid proteins are β₂-microglobulin. Thus, it is assumed that carpal tunnel syndrome iscaused by the deposition of the β₂ -microglobulin on the diseased part,which is accumulated in the blood with high level. Thus, it is expectedthat carpal tunnel syndrome can be prevented by removing the β₂-microglobulin in the blood along with the artificial blood dialysis.Further, it is possible that β₂ -microglobulin is involved in thedeposition of amyloid on the parts other than the carpal tunnel.

Heretofore, no disease has been known of which cause is clarified to bethe β₂ -microglobulin in the blood, and so how to remove the β₂-microglobulin in the blood has not been considered at all.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a method forselectively removing β₂ -microglobulin in the blood.

The object can be accomplished by the present invention which provides acolumn for adsorbing and removing β₂ -microglobulin, which employsimmobilized anti-β₂ -microglobulin antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a) and 1(b) show examples of circuits which employs blooddialyzer and β₂ -microglobulin-removing column together, wherein FIG.1(a) shows an example in which they are connected in series, and FIG.1(b) shows an example in which they are connected in parallel;

FIG. 2 is a schematic view showing the results of SDS-polyacrylamide gelelectrophoresis of column fractions obtained in Example 1;

FIG. 3 shows the total amount of proteins and the level of β₂-microglobulin of the column fractions obtained in Example 2;

FIGS. 4(a), 4(b) and 4(c) show the change in time of the amount of theremaining β₂ -microglobulin in the blood, wherein 4(a) shows the resultsobtained by using a column in which anti-β₂ -microglobulin antibody isimmobilized, 4(b) shows the results obtained by reusing the column of(a), and 4(c) shows the results obtained by using a column in whichanti-β₂ -microglobulin is not immobilized.

BEST MODE FOR CARRYING OUT THE INVENTION

Both of a polyclonal antibody which is obtained by immunizing animalssuch as mice, rats, rabbits, goats and sheep, and a monoclonal antibodyobtained by using a cell hybridization technique can be used as theanti- β₂ -microglobulin antibody in the present invention. β₂-microglobulin to be immunized may be derived from any animal if theobtained antibody can bind the human β₂ -microglobulin. However, for thepromotion of the binding efficiency, β₂ -microglobulin derived fromhuman or monkey, especially human is preferred. A peptide fragmentthereof or a synthetic peptide, which has the same immunogenic ability,may also be used. In order to effectively remove β₂ -microglobulin andnot to affect the function of the blood cells, it is preferred to use amonoclonal antibody which does not bind to the β₂ -microglobulinconstituting the HLA on the cell surfaces, and which binds to the freeβ₂ -microglobulin alone.

The insoluble carrier used in the present invention includes agarose,cellulose, dextran, polyacrylamide and polystyrene derivatives. Thematerial of the insoluble carrier is preferably a hydrophilic materialto which only a small amount of blood components are non-specificallyadsorbed. The carrier may be in the form of beads, fibers or films. Incases where the beads are used, the diameter of the beads is not limitedas long as the β₂ -microglobulin-containing fluid can circulate.However, to reduce the flow resistance, those having a diameter of 50 to3,000 μm, especially 200 to 3,000 μm are preferably used. Further, it ispreferred to use beads which are physically strong and of whichdiameters are not changed so much by a pressure applied thereto.

The binding of the antibody to the insoluble carrier may be conducted bychemically forming covalent bonds therebetween by using a coupling agentsuch as cyanogen bromide and carbodiimide, or using a cross-linkingagent such as glutaraldehyde. It is also possible to promote theadsorbing ability per an amount of antibody by binding the β₂-microglobulin via protein A which is preliminary immobilized to theinsoluble carrier. In this case, however, it is necessary to chemicallycross-link the protein A and the antibody to prevent the escape of theantibody.

The amount of the antibody to be immobilized in the column, and the sizeof the column are not restricted. To obtain a better curing effect, itis preferred that one column can adsorb not less than 50 mg of β₂-microglobulin. Since 1 g of the antibody can adsorb 50 mg to 150 mg ofantigen β₂ -microglobulin, it is necessary that one column have 300 mgor more of the antibody. It should be noted, however, if 2 or morecolumn is used in a treatment, the amount of the antibody per column canbe reduced.

In the treatment, the column for removing β₂ -microglobulin may be usedalone. However, in view of the fact that the major subject patients arethose undergoing artificial blood dialysis, it is preferred to connectthe column in series or in parallel with a blood dialyzer tosimultaneously conduct blood circulation in view of the convenience ofthe operation.

An example in which the column of the present invention is connected tothe blood dialyzer will now be described referring to FIG. 1. An examplein which the column is connected in series to the blood dialyzer isshown in FIG. 1 (a). The blood taken out of the body of a patient entersa blood dialyzer 2 through a blood pump 1, and then dialyzed with adialyzing fluid 4. The blood is then subjected to a treatment to removeβ₂ -microglobulin in a β₂ -microglobulin-removing column 3, and thenreturns to the body of the patient. Although the β₂-microglobulin-removing column 3 is connected after the blood dialyzer 2as shown in FIG. 1 (a), the column may also be connected before theblood dialyzer 2. An example in which the column and the blood dialyzerare connected in parallel will now be described referring to FIG. 1 (b).The blood taken out of the body of a patient is divided into twodirections after passing through the blood pump 1. The blood flowproceeding to one direction enters the blood dialyzer 2 in which theblood is dialyzed with the dialyzing fluid 4. The blood flow proceedingto another direction enters the β₂ -microglobulin-removing column 3 inwhich the β₂ -microglobulin is removed, after being controlled of itsflow rate by an auxiliary pump 5, and gets together with the blood flowfrom the blood dialyzer 2 and returns to the body. In cases where thecolumn is connected in parallel to the blood dialyzer, the β₂-microglobulin-removing column may be connected in any portion of thecircuit. In cases of connecting the column and the blood dialyzer inparallel, to make the flow rate in the bypass constant, the auxiliarypump 5 may be used as shown in FIG. 1 (b). The control of the flow ratemay also be accomplished by appropriately selecting the inner diametersof the tubes of the circuit without using the auxiliary pump 5. Thematerial consitituting the dialysis membrane of the blood dialyzer isnot restricted and includes cellulose, cellulose acetate,polymethylmethacrylate, polyacrylonitrile, polysulphones, polyamides,polyesters, polyvinylalcohols and polyvinylalcohol copolymers. Toincrease the amount of the β₂ -microglobulin removed, it is preferredthat the dialysis membrane have a permeability of 2% or more for theproteins with a molecular weight of 10,000.

Whole blood can be passed through the column for removing β₂-microglobulin of the present invention. Although the operation iscomplicated, the same effect may be obtained by circulating the plasmafrom which the blood cells have been removed by a conventional plasmaseparator, instead of circulating the whole blood.

The column used for adsorption may be regenerated and reused by passingan acidic solution with a pH of about 2 through the used column.

Since the column of the present invention selectively adsorbs β₂-microglobulin, the β₂ -microglobulin in the blood can be convenientlyand effectively removed. Further, the column of the present inventionhas an advantage that it can be repeatedly used by eluting the adsorbedβ₂ -microglobulin.

The present invention will now be described more specifically referringto the examples thereof.

EXAMPLE 1

To 1 ml of an agarose gel ("Affigel 10", manufactured by Bio RadLaboratories) in which N-hydroxysuccinimide ester groups were introducedvia a spacer of 10 atoms length (--OCH₂ CONH(CH₂)NHCO(CH₂)₂ --), 1.46 mgof a commercially available anti-human β₂ -microglobulin monoclonalantibody in 1 ml of 0.lM HEPES-NaOH buffer (pH7.5) was added and themixture was gently stirred overnight.

To the mixture, 0.1 ml of 1M ethanolamine-HCl (pH8.0) was added and themixture was allowed to react for 1.5 hours. After blocking thenon-reacted N-hydroxysuccinimide ester groups, the gel was washedalternately three times with 1 ml of 0.lM acetic acid-NaOH (pH4.0) and 1ml of 0.lM carbonic acid-NaOH (pH9.0), each containing 0.5M NaCl.Finally the gel was equilibrated with PBS. The amount of the remainingprotein after the immobilization was 0.02 mg, and so 1.44 mg of antibodywas immobilized in 1 g of the gel.

In a commercially available small column (0.8 mm of diameter), 0.3 ml ofthe thus obtained antibody-immobilized gel was packed, and a modelsolution containing 0.1 mg/ml of bovine serum albumin (BSA) and 0.1mg/ml of human β₂ -microglobulin in PBS was passed through the column ata flow rate of 2.4 ml/h at room temperature. Upon starting the flow,fractions of 0.63 ml each were recovered by using a fraction collector,and 20 μl aliquotes of each fraction (2-5) were analyzed bySDS-polyacrylamide electrophoresis.

The results are shown in FIG. 2. FIG. 2 is a schematic view showing theresults of the analysis by the electrophoresis. Lane 1 shows the resultobtained by subjecting the model protein solution to the electrophoresisbefore passing through the column, and lanes 2-5 show the results of theelectrophoresis of the fractions 2-5, respectively.

The arrow indicates the migrated position of β₂ -microglobulin (β₂ m)and the reference sample of BSA.

In all of the analyzed fractions 2-5, the amount ratio of the β₂-microglobulin to the BSA is smaller than in the solution before beingsubjected to the column, which shows that only β₂ -microglobulin wasselectively adsorbed to the column.

Further, after the proteins remained in the column was eluted with PBS,the antigen bound to the antibody was eluted with 50 mM glycine-HClbuffer (pH2.4), and only β₂ -microglobulin was eluted. The resultobtained by subjecting the eluted solution to the electrophoresis isshown in FIG. 2, lane 6.

EXAMPLE 2

Through a column prepared in the same manner as in Example 1, a serumcontaining a high level of β₂ -microglobulin from a patient who wasundergoing artificial blood dialysis was passed. From the beginning ofthe passage, fractions of 0.32 ml each were recovered using a fractioncollector.

Total protein amount (expressed in terms of the absorbance at 280 nm)and the level of the β₂ -microglobulin (β₂ m) determined by animmunoassay, of the each fraction are shown in FIG. 3. In the fractionsup to No. 10, the amount ratio of the β₂ -microglobulin to the totalprotein amount is significantly smaller than that of the serum beforebeing subjected to the column, which shows that the β₂ -microglobulinwas adsorbed and removed by the antibody (As for the serum before beingsubjected to the column, the absorbance at 280 nm was 72.1, and the β₂-microglobulin level was 40.5 mg/ml).

It can be seen from the results shown in FIG. 3 that the total amount ofthe β₂ -microglobulin adsorbed to the column was 0.049 mg, and so 0.11mg of β₂ -microglobulin was adsorbed per 1 mg of immobilized antibody.

EXAMPLE 3

Two milligrams of the commercially available anti-human β₂-microglobulin used in Examples 1 and 2 was mixed with 2.8 ml ofcellulose beads ("Formyl-Cellulofine", manufactured by ChissoCorporation) in which formyl groups had been introduced via a spacer of9 atoms length (--OCH₂ CH(OH)CH₂ NH(CH₂)₄ --) in 6 ml of potassiumphosphate buffer (pH7.0). After reacting for 2 hours at 4° C.,dimethylamineborane was added and the reaction was allowed to continueovernight under the reducing condition to prepare beads to which 0.54 mgof antibody was immobilized per 1 ml of carrier. The non-reacted formylgroups were blocked by reacting them with the amino groups of Tris.

In a small column, 2.1 ml (1.1 mg in terms of antibody) of the beadswere packed, and 10 ml of normal human blood to which β₂ -microglobulinwere added was circulated for 2 hours at a flow rate of 1 ml/min. Smallaliquotes of the blood were taken at appropriate times and the β₂-microglobulin (β₂ m) levels thereof were determined. The results areshown in FIG. 4 (a). The adsorption was completed within 10 minutes fromthe commencement of the circulation, and the adsorbed amount was 100 μg,which is about 1/10 of the amount of the antibody used.

After washing the column with 1M glycine-HCl buffer (pH2.8), the samecirculation experiment was repeated. As shown in FIG. 4 (b), same orbetter adsorption than the first circulation was observed. Thus, thecolumn was able to be regenerated. In the control experiment in whichcellulose beads having no antibody immobilized thereto were used, theadsorption was scarecely observed (FIG. 4 (c)).

INDUSTRIAL APPLICABILITY

Since the column of the present invention can specifically adsorb andremove the β₂ -microglobulin in the blood as described above, the columnof the present invention is very helpful for the prevention andtreatment of the complications including amyloidosis such as carpaltunnel syndrome, and osteopathy.

We claim:
 1. A system for dialyzing blood comprising a blood dialyzerand a column for removing β₂ -microglobulin including insoluble carrierand an anti β₂ -microglobulin antibody immobilized to the carrier, whichcolumn is connected in series or in parallel to the blood dialyzer. 2.The system of claim 1 wherein the antibody is a monoclonal antibody.